Functional Assessment of the Medicago truncatula NIP/LATD Protein Demonstrates That It Is a High-Affinity Nitrate Transporter    

Abstract

The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are lowaffinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 mM) concentrations than at higher (5 mM) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1

Nitrate transporters: an overview in legumes

“…In M. truncatula, both MtNPFs studied were expressed and characterized in Xenopus oocytes. The first studied protein, MtNPF1.7, was demonstrated to be a high-affinity nitrate transporter (Bagchi et al 2012). This result indicates that the classification made until now (indicating that low-affinity nitrate transporters belong to the NPF family and highaffinity transporters belong to the NRT2 family) is too simplistic.…”

“…This result indicates that the classification made until now (indicating that low-affinity nitrate transporters belong to the NPF family and highaffinity transporters belong to the NRT2 family) is too simplistic. MtNPF1.7 can restore chlorate sensitivity in resistant mutants of A. thaliana, but is not involved in the nitrate uptake in M. truncatula (Bagchi et al 2012;Salehin et al 2013). The second studied protein, MtNPF6.8, has the proprieties of a dual-affinity nitrate transporter when expressed in Xenopus oocytes (Morère-Le Paven et al 2011).…”

“…The knockdown of MtNPF6.8 affected the entire inducible component of the LATS, suggesting that MtNPF6.8 is an essential actor of the inducible LATS (Pellizzaro et al 2014). So, to date, both studied nitrate transporters from M. truncatula, MtNPF6.8 and MtNPF1.7, can transport nitrate at low concentrations when expressed in heterologous transport system, without having a HATS activity in planta (Morère-Le Paven et al 2011; Bagchi et al 2012;Pellizzaro et al 2014). Interestingly, AtNPF6.3 of A. thaliana, known to be a dual-affinity nitrate transporter in oocytes, has been shown to be involved in HATS in planta (Wang et al 1998;Liu et al 1999;Ho et al 2009), although this is a matter of debate (Glass and Kotur 2013).…”

“…In M. truncatula, studies (summarized on Fig. 4) have shown that both characterized nitrate transporters, MtNPF1.7 and MtNPF6.8, play a role in root development Bagchi et al 2012;Pellizzaro et al 2014). MtNPF1.7 is expressed in the tips of primary and lateral roots (including the root cap, meristem and proximal portion of the elongation zone) as well as in the nodule meristem (Fig.…”

“…In M. truncatula, it has been shown that MtNPF1.7 is essential in the formation and maintenance of nodule meristems and in rhizobial invasion during nodulation (Veereshlingam et al 2004;Harris and Dickstein 2010). Using different mutants of MtNPF1.7, Bagchi et al (2012) proposed that the ability of MtNPF1.7 to transport nitrate is correlated with the abilities of mutants to form and maintain nodules. However, these same authors also proposed that MtNPF1.7 should have another unknown activity, besides nitrate transport, in nodule development and in root architecture.…”